Opposing effects of the angiopoietins on the thrombin-induced permeability of human pulmonary microvascular endothelial cells

Abstract

Background: Angiopoietin-2 (Ang-2) is associated with lung injury in ALI/ARDS. As endothelial activation by thrombin plays a role in the permeability of acute lung injury and Ang-2 may modulate the kinetics of thrombin-induced permeability by impairing the organization of vascular endothelial (VE-)cadherin, and affecting small Rho GTPases in human pulmonary microvascular endothelial cells (HPMVECs), we hypothesized that Ang-2 acts as a sensitizer of thrombin-induced hyperpermeability of HPMVECs, opposed by Ang-1.

Methodology/principal findings: Permeability was assessed by measuring macromolecule passage and transendothelial electrical resistance (TEER). Angiopoietins did not affect basal permeability. Nevertheless, they had opposing effects on the thrombin-induced permeability, in particular in the initial phase. Ang-2 enhanced the initial permeability increase (passage, P = 0.010; TEER, P = 0.021) in parallel with impairment of VE-cadherin organization without affecting VE-cadherin Tyr685 phosphorylation or increasing RhoA activity. Ang-2 also increased intercellular gap formation. Ang-1 preincubation increased Rac1 activity, enforced the VE-cadherin organization, reduced the initial thrombin-induced permeability (TEER, P = 0.027), while Rac1 activity simultaneously normalized, and reduced RhoA activity at 15 min thrombin exposure (P = 0.039), but not at earlier time points. The simultaneous presence of Ang-2 largely prevented the effect of Ang-1 on TEER and macromolecule passage.

Conclusions/significance: Ang-1 attenuated thrombin-induced permeability, which involved initial Rac1 activation-enforced cell-cell junctions, and later RhoA inhibition. In addition to antagonizing Ang-1, Ang-2 had also a direct effect itself. Ang-2 sensitized the initial thrombin-induced permeability accompanied by destabilization of VE-cadherin junctions and increased gap formation, in the absence of increased RhoA activity.

Figure 1. Effect of angiopoietins (Ang) on basal permeability, Tie2 phosphorylation and RhoA activity of human pulmonary microvascular endothelial cells (HPMVECs).

Data are presented as mean ± standard error of the mean. NS: not significant. a. Neither Ang-2, nor Ang-1 affected the basal horse radish peroxidase (HRP) passage as measured 30 min after their addition (n = 7–9). b. Neither Ang-2, nor Ang-1 affected the basal transendothelial electrical resistance (TEER) as measured 30 min after their addition (n = 9–17). c. Representative western blots of pTie2, tTie and immunoprecipitated (IP) tyrosine phosphorylated (pTyr) and total Tie2 are shown. d. Ang-1 induced a transient increase in the phosphorylated Tie2 (pTie2)/total Tie2 (tTie2) ratio, with a maximum at 15 and 30 min after its addition (*P = 0.0046, **P = 0.0003). Ang-2 blocked the Ang-1-induced increase in Tie2 phosphorylation at 15 and 30 min (#P = 0.0073, ##P = 0.0134, n = 5–9). Ang-2 alone did not affect Y1100 Tie2 phosphorylation.

Figure 2. Effect of angiopoietins (Ang) on initial and prolonged permeability of human pulmonary microvascular endothelial cells (HPMVECs) during thrombin stimulation.

Data are presented as mean ± standard error of the mean. NS: not significant. a. An averaged curve of horse radish peroxidase (HRP) passage of control cells stimulated with thrombin (IIa) at time = 0 min, showing the typical s-shape (n = 9). Cells were stimulated with angiopoietins (A) at time = −30 min. The initial HRP passage rate from time = 0–15 min and the prolonged HRP passage rate from time = 30–90 min were calculated as indicated by the vertical dashed lines. b. Representative experiment (in triplo) showing averaged curves of HRP passage of control and angiopoietin-stimulated cells. Cells were stimulated with angiopoietins at time = −30 min and with thrombin at time = 0 min. c. Ang-2 increased the initial HRP passage rate (*P = 0.010), while Ang-1 or the combination did not affect it (n = 7–9). d. Neither Ang-2, nor Ang-1 affected the prolonged HRP passage rate (n = 8–9). e. An averaged curve of the transendothelial electrical resistance (TEER) of control cells stimulated with thrombin at time = 0 min (n = 17). Cells were stimulated with angiopoietins at time = -30 min. The initial rate of TEER decrease from time = 0–10 min and the prolonged maximum TEER decrease were calculated as indicated by the vertical dashed lines. f. Representative experiment (in triplo) showing averaged curves of the TEER of control and angiopoietin-stimulated cells. Cells were stimulated with angiopoietins at time = −30 min and with thrombin at time = 0 min. g. Ang-2 and the combination enhanced the initial rate of the TEER decrease (**P = 0.021, ^#P = 0.036), while Ang-1 reduced it (*P = 0.027, n = 12–16). h. Ang-2 or the combination did not affect the prolonged maximum TEER decrease, while Ang-1 reduced it (*P<0.0001, n = 12–16).

Figure 3. a, b and c. Effect of angiopoietins on the molecular organization of vascular endothelial cadherin (VE-cadherin) in human pulmonary microvascular endothelial cells (HPMVECs).

Immunofluorescence morphological analysis and quantification of the distribution of VE-cadherin in control cells and cells treated with angiopoietins (Ang) and thrombin (IIa) for 2 (a) and 15 (b) min. Cells were stained with antibodies specific for VE-cadherin (green), with rhodamin-phalloidin for actin filaments (F-actin, red) and with 4′6-diamidino-2-phenylindole (DAPI) for the nuclei (blue). A 63x magnification is shown. The arrows indicate intercellular gaps. The left panel shows the VE-cadherin staining at the cell-cell borders. The middle panel shows the F-actin cytoskeleton. The right panel shows the merge of VE-cadherin, F-actin and the nuclei. a. The thrombin response after 2 min. b. The thrombin response after 15 min. c. Ang-2 enhances (*P<0.0001) and Ang-1 reduces (**P = 0.0257) the formation of thrombin-induced interendothelial gaps (*P<0.0001 thrombin stimulation vs. control). Total gap area was determined as described in the methods section. Data are the mean ± standard error of the mean from at least 6 pictures per condition.

Figure 4. Effect of angiopoietins (Ang) on Rac1 activity of thrombin (IIa)-stimulated human pulmonary microvascular endothelial cells (HPMVECs).

Data are presented as mean ± standard error of the mean. a. Thrombin stimulation reduced Rac1 activity 1 min after stimulation (*P = 0.0256), independent of angiopoietin treatment (n = 5–6). b. Rac1 activity was normalized 15 min after thrombin stimulation, independent of angiopoietin treatment (n = 5–6).

Figure 5. Effect of angiopoietins (Ang) on RhoA activity of thrombin (IIa)-stimulated human pulmonary microvascular endothelial cells (HPMVECs).

Data are presented as mean ± standard error of the mean. NS: not significant. a. RhoA activity was increased 1 min after thrombin addition (*P<0.0001), independent of angiopoietin treatment (n = 7–8). b. RhoA activity was still increased 15 min after thrombin addition (*P<0.0001), but the increase became less in cells treated with Ang-1 (**P = 0.039, n = 6) or the combination (***P = 0.034, n = 6-8).